Small micromeres contribute to the germline in the sea urchin

Many indirect developing animals create specialized multipotent cells in early development to construct the adult body and perhaps to hold the fate of the primordial germ cells. In sea urchin embryos, small micromeres formed at the fifth division appear to be such multipotent cells: they are relatively quiescent in embryos, but contribute significantly to the coelomic sacs of the larvae, from which the major tissues of the adult rudiment are derived. These cells appear to be regulated by a conserved gene set that includes the classic germline lineage genes vasa, nanos and piwi. In vivo lineage mapping of the cells awaits genetic manipulation of the lineage, but previous research has demonstrated that the germline is not specified at the fourth division because animals are fertile even when micromeres, the parent blastomeres of small micromeres, are deleted. Here, we have deleted small micromeres at the fifth division and have raised the resultant larvae to maturity. These embryos developed normally and did not overexpress Vasa, as did embryos from a micromere deletion, implying the compensatory gene regulatory network was not activated in small micromere-deleted embryos. Adults from control and micromere-deleted embryos developed gonads and visible gametes, whereas small micromere-deleted animals formed small gonads that lacked gametes. Quantitative PCR results indicate that small micromere-deleted animals produce background levels of germ cell products, but not specifically eggs or sperm. These results suggest that germline specification depends on the small micromeres, either directly as lineage products, or indirectly by signaling mechanisms emanating from the small micromeres or their descendants.     

A Stereometric Analysis of Karyokinesis, Cytokinesis and Cell Arrangements during and following Fourth Cleavage Period in the Sea Urchin, Lytechinus variegatus

Fourth cleavage of the sea urchin embryo produces 16 blastomeres that are the starting point for analyses of cell lineages and bilateral symmetry. We used optical sectioning, scanning electron microscopy and analytical 3-D reconstructions to obtain stereo images of patterns of karyokinesis and cell arrangements between 4th and 6th cleavage. At 4th cleavage, 8 mesomeres result from a variant, oblique cleavage of the animal quartet with the mesomeres arranged in a staggered, offset pattern and not a planar ring. This oblique, non-radial cleavage pattern and polygonal packing of cells persists in the animal hemisphere throughout the cleavage period. Contrarily, at 4th cleavage, the 4 vegetal quartet nuclei migrate toward the vegetal pole during interphase; mitosis and cytokinesis are latitudinal and subequatorial. The 4 macromeres and 4 micromeres form before the animal quartet divides to produce a 12-cell stage. Subsequently, macromeres and their derivatives divide synchronously and radially through 8th cleavage according to the Sachs-Hertwig rule. At 5th cleavage, mesomeres and macromeres divide first; then the micromeres divide latitudinally and unequally to form the small and large micromeres. This temporal sequence produces 28-and 32-cell stages. At 6th cleavage, macromere and mesomere descendants divide synchronously before the 4 large micromeres divide parasynchronously to produce 56- and 60-cell stages.     

Programmed reduction of ABC transporter activity in sea urchin germline progenitors

ATP-binding cassette (ABC) transporters protect embryos and stem cells from mutagens and pump morphogens that control cell fate and migration. In this study, we measured dynamics of ABC transporter activity during formation of sea urchin embryonic cells necessary for the production of gametes, termed the small micromeres. Unexpectedly, we found small micromeres accumulate 2.32 times more of the ABC transporter substrates calcein-AM, CellTrace RedOrange, BoDipy-verapamil and BoDipy-vinblastine, than any other cell in the embryo, indicating a reduction in multidrug efflux activity. The reduction in small micromere ABC transporter activity is mediated by a pulse of endocytosis occurring 20-60 minutes after the appearance of the micromeres--the precursors of the small micromeres. Treating embryos with phenylarsine oxide, an inhibitor of endocytosis, prevents the reduction of transporter activity. Tetramethylrhodamine dextran and cholera toxin B uptake experiments indicate that micromeres have higher rates of bulk and raft-associated membrane endocytosis during the window of transporter downregulation. We hypothesized that this loss of efflux transport could be required for the detection of developmental signaling molecules such as germ cell chemoattractants. Consistent with this hypothesis, we found that the inhibition of ABCB and ABCC-types of efflux transporters disrupts the ordered distribution of small micromeres to the left and right coelomic pouches. These results point to tradeoffs between signaling and the protective functions of the transporters.     

Identification and characterization of bone morphogenetic protein 2/4 gene from the starfish Archaster typicus

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.     

Nanos functions to maintain the fate of the small micromere lineage in the sea urchin embryo

The translational regulator nanos is required for the survival and maintenance of primordial germ cells during embryogenesis. Three nanos homologs are present in the genome of the sea urchin Strongylocentrotus purpuratus, all of which are expressed with different timing in the small micromere lineage. This lineage is set-aside during embryogenesis and contributes to constructing the adult rudiment. Small micromeres lacking Sp-nanos1 and Sp-nanos2 undergo an extra division and are not incorporated into the coelomic pouches. Further, these cells do not accumulate Vasa protein even though they retain vasa mRNA. Larvae that develop from Sp-nanos1 and 2 knockdown embryos initially appear normal, but do not develop adult rudiments; although they are capable of eating, over time they fail to grow and eventually die. We conclude that the acquisition and maintenance of multipotency in the small micromere lineage requires nanos, which may function in part by repressing the cell cycle and regulating other multipotency factors such as vasa. This work, in combination with other recent results in Ilyanassa and Platynereis dumerilii, suggests the presence of a conserved molecular program underlying both primordial germ cell and multipotent cell specification and maintenance.     

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.     

The pattern of cell division in the early development of the sea urchin,Paracentrotus lividus

In the sea urchin Paracentrotus lividus, the first three cleavages are synchronous in all blastomeres. Upon the segregation of the micromeres at the fourth cleavage, a vegetal-animal gradient of cell division begins; i.e., the closer the cells are to the micromeres, the earlier they enter mitosis. The phase difference between mitotic cells along the vegetal-animal axis increases concurrently with the increase in cell number. At the blastula stage, mitoses appear organized in clusters. From the mesenchyme-blastula stage onward, the clusters become smaller and most of the mitotic cells are solitary. The size of the clusters increases upon treatment with colchicine, thus suggesting the existence of pacemaker units of mitotic activity. We confirm that the division of the micromeres is out of phase with respect to the other blastomeres. Of the eight cells originating from the first division of the micromeres, only the four outer ones continue to cleave; the four inner ones appear to have lost the ability to divide. The overall mitotic activity is high during cleavage and suddenly drops to very low levels around hatching. An interpretation of these results is presented in terms of a nonlinear oscillators theory.     

Mass isolation and culture of sea urchin micromeres

Summary A procedure is described for large-scale isolation of micromeres from 16-cell stage sea urchin embryos. One to two grams of >99% pure, viable micromeres (2.3 to 4.6 × 10⁸ cells) are routinely isolated in a single preparation. In culture, these cells uniformly proceed through their normal development, in synchrony with micromeres in whole embryos, ultimately differentiating typical larval skeletal structures. The attributes of this procedure are: (a) the very early time of isolation of the cells, directly after the division that establishes the cell line; (b) the large yield of cells; (c) the purity of the preparation of cell; and (d) their synchronous development in culture through skeletogenesis. The procedure greatly aids in making sea urchin micromeres a favorable material for molecular analysis of development. This work was supported in part by the following grants from the National Institutes of Health: Grant HL-10312 to A.H.W., Grant GM-20784 to Helen R. Whiteley, Grant ES-02190 to N. Karle Mottet, M.D., and Training Grants ES-07032 and HD-00266.     

Nuclear beta-catenin-dependent Wnt8 signaling in vegetal cells of the early sea urchin embryo regulates gastrulation and differentiation of endoderm and mesodermal cell lineages

The entry of beta-catenin into vegetal cell nuclei beginning at the 16-cell stage is one of the earliest known molecular asymmetries seen along the animal-vegetal axis in the sea urchin embryo. Nuclear beta-catenin activates a vegetal signaling cascade that mediates micromere specification and specification of the endomesoderm in the remaining cells of the vegetal half of the embryo. Only a few potential target genes of nuclear beta-catenin have been functionally analyzed in the sea urchin embryo. Here, we show that SpWnt8, a Wnt8 homolog from Strongylocentrotus purpuratus, is zygotically activated specifically in 16-cell-stage micromeres in a nuclear beta-catenin-dependent manner, and its expression remains restricted to the micromeres until the 60-cell stage. At the late 60-cell stage nuclear beta-catenin-dependent SpWnt8 expression expands to the veg2 cell tier. SpWnt8 is the only signaling molecule thus far identified with expression localized to the 16-60-cell stage micromeres and the veg2 tier. Overexpression of SpWnt8 by mRNA microinjection produced embryos with multiple invagination sites and showed that, consistent with its localization, SpWnt8 is a strong inducer of endoderm. Blocking SpWnt8 function using SpWnt8 morpholino antisense oligonucleotides produced embryos that formed micromeres that could transmit the early endomesoderm-inducing signal, but these cells failed to differentiate as primary mesenchyme cells. SpWnt8-morpholino embryos also did not form endoderm, or secondary mesenchyme-derived pigment and muscle cells, indicating a role for SpWnt8 in gastrulation and in the differentiation of endomesodermal lineages. These results establish SpWnt8 as a critical component of the endomesoderm regulatory network in the sea urchin embryo.     

Complete regulation of development throughout metamorphosis of sea urchin embryos devoid of macromeres

The developmental potential of the animal cap (consisting of eight mesomeres) recombined with micromeres or of micromere progeny was examined in sea urchin embryos. The embryos derived from the animal cap recombined with a quartet of micromeres or their descendants developed into four-armed plutei. After feeding, the larvae developed into eight-armed plutei. The left-right polarity of the larvae, recognized by the location of the echinus rudiment, was essentially normal, regardless of the orientation of animal-vegetal polarity in micromeres combining with the animal cap. The larvae had sufficient potential to metamorphose into complete juvenile sea urchins with five-fold radial symmetry. Cell lineage tracing experiments showed that: (i) macromere progeny were not required for formation of the typical pattern of primary mesenchyme cells derived exclusively from large micromeres; (ii) the progeny of large micromeres did not contribute to cells in the endodermal gut with three compartments of normal function; (iii) the presumptive ectoderm had the potential to differentiate into endodermal gut and mesodermal secondary mesenchyme cells, from which pigment cells likely differentiated; and (iv) behavior of the progeny of small micromeres was the same as that in normal embryos through the gastrula stage. These results indicate that the mesomeres respecify their fate under the inductive influence of micromeres so perfectly that complete juvenile sea urchins are produced.     

The synthesis and secretion of collagen by cultured sea urchin micromeres

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.     

Unequal divisions at the third cleavage increase the number of primary mesenchyme cells in sea urchin embryos

To clarify the distribution and behavior of the maternal factors that direct the differentiation of primary mesenchyme cells (PMC) in sea urchin embryos, unequal division was induced at the third cleavage with the treatment of dinitro-phenol (DNP), and the numbers of differentiated PMC were examined. The most surprising finding was that the number of PMC was considerably increased in some of the DNP-treated embryos. This increase in the number of PMC was suggested to be closely related to the size of the precocious micromeres formed at the 8-cell stage. By measuring both the size of the precocious micromeres and the number of PMC in individual embryos, it was suggested that almost all the descendants of the precocious micromeres differentiated into PMC, if the volume was less than 26 pL (about three times the volume of normal micromeres). Cell tracing experiments ascertained that precocious micromeres with small volumes behave just like micromeres formed at the fourth cleavage in normal embryos. The obtained results indicated that the maternal factors present in sea urchin embryos can direct, at least, more than three times the number of PMC, and that the number of cell divisions of the PMC lineage is not strictly regulated.     

Cyclin E and its associated cdk activity do not cycle during early embryogenesis of the sea Urchin

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Strongylocentrotus purpuratus. Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity.     

STUDIES ON UNEQUAL CLEAVAGE IN SEA URCHINS I. MIGRATION OF THE NUCLEI TO THE VEGETAL POLE

It has been known for nearly a century that at the 16-cell stage of sea urchin embryos, the animal 4 cells divide equally and horizontally, whereas the vegetal 4 cells cleave unequally and practically vertically into macromeres and micromeres. Recently, more careful observations were made on the process of micromere formation and it has been revealed that a primary cause for the inequality lies in the migration of the 4 vegetal nuclei to the vegetal pole of the embryo which brings about excentricity of the mitotic apparatus. Records of this phenomenon are given in the present paper.     

Synthesis of RNA by dissociated cells of the sea urchin embryo

The incorporation of radioactive uridine into RNA by micromeres, mesomeres and macromeres of sea urchin embryos was studied, employing methods for separating the cell types in pure suspension. At the 16-cell stage, the 3-cell types, on a per genome basis, synthesized RNA at approximately the same rate although on a per mg protein basis the micromere-RNA synthetic rate was considerably higher than either mesomeres or macromeres. At the 32-cell stage, incorporation of radioactive uridine by micromeres decreased relative to mesomeres and macromeres. It was demonstrated that radioactive uridine could not be effectively washed or diluted out of the cells of 16-cell stage embryos. Experiments on reaggregating cells did not detect any transfer or transport of radioactivity from micromeres to the other cells. Possible explanations for these findings versus the disparate results of previous investigators were presented.     

Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development.     

Studies on Unequal Cleavage in Sea Urchins III. Micromere Formation under Compression

When sea urchin embryos at 2-cell stage are flattered between agar plates, the direction of cleavage is rotated by 90° in each division in reference to the preceding cleavage and no micromere is formed. But under this condition, micromeres are formed in 2 cases; 1) When the egg axis is parallel to the plane of flattening, 2 micromeres are formed on one side of a square 16-cell stage. 2) when the egg axis is perpendicular to the plane, 4 micromeres are formed at the center of the square.
When put into a groove, a string of 4 cells is formed showing that the spindle direction is further deflected by the groove. In the following 16-cell stage in the groove, which consists of 2 layers of 8 cells, cases with 2 micromeres on one side and 4 micromeres at the center are still found. If the 2-cell stage is introduced into a groove after the formation of mitotic apparatus, the spindle direction can no longer be changed and the 4-cell stage becomes like 4 pancakes stuck in 2 layers, indicating that 2 asters are holding the ends of a spindle in fixed positions.